MackenzieDepartment of Bio

Character cauda-epid

ization of male mouse idymal -L-fucosidaseJ. Bartlett, Som Phopin, Barry Beanlogy, Lehigh University, Bethlehem PA 18015

Background• Semen specific α-L fucosidase forms in rats, Unio

elongatulus, bulls, and humans (3)(4)(5)(6)

• Carbohydrate involvement in sperm-egg interaction(1)

• Fucose-binding protein a potential receptor on sperm membrane(2)

• α-L fucosidase mediated fertilization in Halocynthia roretzi (7)

• Majority α-L fucosidase in human seminal plasma(8)

• Capacitated sperm immunolocalize strongly at anterior head, faintly at midpiece and some tails(9)

• Acrosome-reacted sperm label in equatorial region(9)

AbstractCharacterization of the -L-fucosidase enzyme in male

mice was performed on cauda epididymal contents(CEC), containing mature sperm and caudal fluid. The enzyme has been implicated as playing a central role in fertilization. To fully understand the role of -L-fucosidase, we have decided to employ a mouse model. The mouse model will allow us to carry out experiments not possible in humans. Here we report on the distribution, subcellular crypticity, and inhibition profiles with two different inhibitors. Information on the inhibitors’ action will allow us to make comparable observations about the effects of these on fertilization. Most -L-fucosidase activity is found in the CEC supernatant. This source is shown to be significantly inhibited by both 18a and DFJ inhibitors. A large amount of cryptic -L-fucosidase activity is released to the supernatant after capacitation. Permeabilization with Triton-X also shows crypticity of uncapacitated and capacitated cells. Ultimately, a characterization of -L-fucosidase will further our understanding of the molecular mechanisms involved in mammalian fertilization.

Experimental Procedure• Postmortem retrieval cauda epididymis and vas deferens

• Incubate minced tissue at 37˚C, 5% CO2 in 1ml HSM to release CEC

• Centrifuge CEC at 500 X g, and remove supernatant

• Wash cells twice and resuspend in HSM= uncapacitated cells

http://evolution.knu.ac.kr/en/contents/upload/001-male.jpg

http://ocw.mit.edu/NR/rdonlyres/Biological-Engineering/20-109Fall-2007/A085142C-2DB3-41EA-B05A-2DC613F0AD9A/0/removing_cells.jpg

Supernatant

Sperm Cell Pellet

4-Mu - Fucose α-L-Fucosidase

Fucose + 4-Mu

• α-L-fucosidase Enzyme Assay with Fluorogenic Substrate-Mix substrate 4-Mu-FUC with HSM

-Add sample at time 0, measure fluorescence over time

-Data are expressed as enzyme activity (slope of fluorescence over time)• As a % of the control, or untreated sample

Fig 1: This graph shows enzyme distribution in CEC. Cell loss during washing stages is responsible for the loss of activity from CEC to Supernatant + Uncapacitated Pellet. P values for supernatant and uncapacitated pellet are 0.0194 and 0.0001 respectively over 5 replicates.

CEC Sup Uncap0

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Distribution of Enzyme In CEC

Distribution of Enzyme within CEC

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Results

Fig 2: This figure compares the inhibition of enzyme activity in mouse CEC supernatant with concentrations of treatment with 18a and DFJ. Both inhibitors show significant inhibition. P values are <0.01 for significant values, over 3-6 replicates*DFJ inhibits Human seminal α-L-fucosidase. Preliminary data suggests that 18a also inhibits Human seminal α-L-fucosidase. Inhibitor 18a was a gift from Dr. Inmaculada Robina.(10)

0M 20mM 10mM 1mM 100µM 1µM 100nM 10nM-5

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Inhibition of Mouse CEC Supernatant with 18a and DFJ

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Inhibition• Treat supernatant for 10 minutes with concentrations

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Capacitation• Treat uncapacitated cells with 1.5% BSA for 1 hour,

37˚C, 5% CO2

Image: Venditti et al. show the change

in staining after sperm cell

capacitation(9).

A- Washed Uncapacitated cellsB- Capacitated cells

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Fig 3: Cell suspensions show a significant increase in activity after capacitation of the uncapacitated pellet. The capacitated supernatant contains the majority of enzyme activity after capacitation. P values are 0.0034 and 0.0499 respectively over 11 replicates.

Permeabilization• Treat cells with various concentrations of

Triton-X

Fig 4: Treatments with Triton-X show a decrease in activity at high concentrations. Uncapacitated cells show increased activity with 0.001% TX, whereas Capacitated cells show the most activity at 0.01% TX, with a small increase in activity at 0.001% TX. No significant change in enzyme activity is seen when supernatant is treated with TX(data not shown). P values are <0.05. Data is from >4 replicates.

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Permeabilization of Cells with Triton-X

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Conclusions

• ~80% CEC α-L-fucosidase activity is in the soluble fraction. ~10% enzyme remains associated with the sperm cell.

• Mouse supernatant α-L-fucosidase enzyme can be significantly inhibited by either 18a or DFJ inhibitors.

• Capacitation shows crypticity- sperm contain more enzyme than is detectable in uncapacitated cells. Most is solubilized.

• Permeabilization of uncapacitated and capacitated cells shows cryptic increase in enzyme activity.

Future Experiments

• Determine enzyme localization after acrosome reaction.

• Quantify α-L-fucosidase activity per sperm cell.

• Characterize stability of α-L-fucosidase over time.

• Stain mouse sperm cells at unwashed, capacitated, and acrosome reacted cells with anti-α-L-fucosidase antibodies.

• Test effects of fluorous surfactants on enzyme activity of mouse sperm.

• Characterize α-L-fucosidase in the female genital tract, uterus, and oocytes.

Acknowledgements

1. Jauhiainen A and Vanha-Pertula T. 1986. α-L-Fucosidase in the preproductive organs and seminal plasma of the bull. Biochem Biophys Acta 880:91-95.

2. Focarelli R, Cacace MG, Serglia R, Rosati F. 1997. A nonglycosylated, 68-kDa α-L-fucosidase is bound to the mollusc bivalve Unio elongatulus sperm plasma membrane and differs from a glycosylated 56-kDa form present in the seminal fluid. Biochem Biophys Res Commun 234:54-58.

3. Avilés M, Abascal I, Martinez-Menarguez JA, Castells MT, Skalaban SR, Ballesta J, Alhadeff JA. 1996. Immunocytochemical localization and biochemical characterization of a novel plasma membrane-associated, neutral pH optimum α-L-fucosidase from rat testis and epididymal spermatozoa. Biochem J 318:821-831.

4. Alhadeff JA,Khunsook S, Choowongkomon K, Baney T, Heredia V, Tweedie A, Bean B. 1999. Characterization of human semen α-L-fucosidases. Mol Hum Reprod 5:809-815.

5. Benoff S. 1997. Carbohydrates and fertilization: An overview. Mol Hum Reprod 3:599-637.6. Huang TTF, Ohzu E, Yanagimachi R. 1982. Evidence suggesting that L-Fucose is part of a recognition signal for sperm-zona

pellucida attachment in mammals. Gamete Res 5:355-361.7. Matsumoto M, Hirata J, Hirohashi N, Hoshi M. 2002. Sperm-egg binding mediated by sperm alpha-L-fucosidase in the

ascidian Halocynthia roretzi. Zoolog Sci 19:43-48.8. Khunsook S, Alhadeff JA, Bean BS. 2002. Purification and characterization of human seminal plasma α-L-fucosidase. Mol

Hum Reprod 8:221-227.9. Venditti JJ, Donigan KA, Bean BS. 2007. Crypticity and functional distribution of the membrane associated α-L-fucosidase of

human sperm. Mol Reprod Devel 74:758-766.10. Moreno-Clavijo E, Carmona AT, Vera-Ayoso Y, Moreno-Vargas AJ, Bello C,Vogel P, Robina I. 2009. Synthesis of novel

pyrrolidine 3,4-diol derivatives as inhibitors of α-L-fucosidases. Org. Biomol. Chemistry, 7:1192-1202.

I’d like to thank Dr. Barry Bean, Som Phopin, Wutigri Nimlamool, Elijah Douglass, Nevada Heft and Jackie Taroni

References

0% TX 0.1% TX 0.01% TX 0.001% TX0

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Mouse Supernatant Treatment with Triton-X

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Fig 6: Supernatant shows no significant difference in supernatant activity after treatment with Triton-x.

Other Data

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Inhibition of Human Sem-inal Plasma with 18a

Concentration of 18a during pre-treatment

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Fig 5: Preliminary data showing that 18a works to inhibit seminal α-L-fucosidase in humans